Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase

TY - JOUR

T1 - Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase

AU - Christiansen, Camilla

AU - St Hilaire, Phaedria M

AU - Winther, Jakob R.

PY - 2004

Y1 - 2004

N2 - In eukaryotic cells the enzyme protein disulfide isomerase (PDI) is responsible for the formation and reshuffling of disulfide bonds in secretory proteins. The reaction carried out by PDI involves interaction with a highly complex mixture of polypeptide molecules that are in the process of folding. This means that PDI activity is typically measured in the context of a globular protein folding pathway. The absence of small, well-defined substrates for the quantitation of both oxidation and reduction reactions constitutes an inherent problem in the analysis of PDI activity. We describe a new type of substrate for PDI where two cysteine-containing oligopeptides are connected by an onameric ethylene glycol linker. We term such hybrid compounds PEGtides. The oligopeptides are each marked with a fluorescent aminobenzoic acid and a quenching nitrotyrosine group, respectively. The reversible formation of an intramolecular disulfide bond between fluorophore-containing and quencher-containing peptide segments results in a redox-dependent fluorescence signal. We find a model compound of this type to be a highly sensitive substrate for PDI both in oxidation and in reduction assays under steady state conditions. These aspects should make substrates of this type generally applicable for assaying PDI and other thiol-disulfide exchange enzymes.

AB - In eukaryotic cells the enzyme protein disulfide isomerase (PDI) is responsible for the formation and reshuffling of disulfide bonds in secretory proteins. The reaction carried out by PDI involves interaction with a highly complex mixture of polypeptide molecules that are in the process of folding. This means that PDI activity is typically measured in the context of a globular protein folding pathway. The absence of small, well-defined substrates for the quantitation of both oxidation and reduction reactions constitutes an inherent problem in the analysis of PDI activity. We describe a new type of substrate for PDI where two cysteine-containing oligopeptides are connected by an onameric ethylene glycol linker. We term such hybrid compounds PEGtides. The oligopeptides are each marked with a fluorescent aminobenzoic acid and a quenching nitrotyrosine group, respectively. The reversible formation of an intramolecular disulfide bond between fluorophore-containing and quencher-containing peptide segments results in a redox-dependent fluorescence signal. We find a model compound of this type to be a highly sensitive substrate for PDI both in oxidation and in reduction assays under steady state conditions. These aspects should make substrates of this type generally applicable for assaying PDI and other thiol-disulfide exchange enzymes.

KW - Disulfides

KW - Glutathione

KW - Humans

KW - Kinetics

KW - Oligopeptides

KW - Oxidation-Reduction

KW - Polyethylene Glycols

KW - Protein Disulfide-Isomerases

KW - Substrate Specificity

U2 - 10.1016/j.ab.2004.06.027

DO - 10.1016/j.ab.2004.06.027

M3 - Journal article

C2 - 15351291

SN - 0003-2697

VL - 333

SP - 148

EP - 155

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -